Molecular Mechanisms of the Human S-phase DNA Damage Checkpoint The long-term goal is to understand the molecular mechanism of human S-phase DNA damage checkpoint. Since many tumor suppressor genes are required for the S-phase checkpoint, deregulation of the S-phase checkpoint is important for cancer development. One pathway for the activation of S-phase checkpoint is ATM/CHK2/CDC25A, in which ATM/CHK2 regulates the degradation of the cell cycle regulator CDC25A through the poly-ubiquitination dependent proteolysis pathway in response to ionizing irradiation (IR). ATM also phosphorylates NBS1 in response to IR to activate the S-phase checkpoint. The relationship between these two pathways is not known. The key to the understanding of the human S-phase checkpoint is the identification of downstream effectors and the delineation of molecular mechanism by which the downstream effectors communicate to the cell cycle and DNA replication machinery to execute the checkpoint response. Structural maintenance of chromosomes proteins (SMC1 and SMC3) are evolutionarily conserved chromosomal proteins that are components of the cohesin complex, necessary for sister chromatid cohesion. These proteins may also function in DNA repair. We have established that SMC1 is an effector in the ATM/NBS1 dependent S-phase checkpoint pathway. ATM phosphorylates SMC1 in a NBS1 dependent manner and phosphorylation of SMC1 is required the S-phase checkpoint. The identification of a downstream effector in the ATM/NBS1 pathway creates an opportunity to delineate the molecular mechanism for the human S-phase checkpoint. We propose the following specific aims: (1) Functional analysis of SMC1/3 phosphorylation in response to DNA damage, (2) Identification of upstream elements that are required and the molecular mechanism how ATM phosphorylates SMCI/3, and (3) Delineation of the downstream molecular mechanism by which phosphorylation of SMCI/3 is required for the activation of S-phase checkpoint.